Dna encoding a fibronectin binding protein as well as its preparation

ABSTRACT

The present invention relates to a new recombinant hybrid-DNA-molecule comprising a nucleotide sequence from S. aureus coding for a protein, or polypeptide, having fibronectin binding properties.

TECHNICAL FIELD

The present invention relates to a fibronectin binding protein as well as hybrid-DNA-molecules, e.g. plasmids or phages comprising a nucleotide sequence coding for said protein. Further the invention relates to microorganisms comprising said molecules and their use producing said protein, as well as the synthetic preparation of said protein.

The object of the present invention is to obtain a minimal fibronectin binding protein.

A further object is to obtain said protein by means of a genetic engineering technique by using e.g. a plasmid comprising a nucleotide sequence coding for said protein.

A further object is to obtain a possibility of preparing said protein by chemical synthesis.

Further objects will be apparent from the following description.

BACKGROUND OF THE INVENTION

WO-A1-85/05553 discloses bacterial cell surface proteins having fibronectin, fibrinogen, collagen, and/or laminin binding ability. Thereby it is shown that different bacteria have an ability to bind to fibronectin, fibrinogen, collagen, and/or laminin. It is further shown that fibronectin binding protein has a molecular weight of 165 kD and/or 87 kD, whereby it is probable that the smaller protein is a part of the larger one.

Fibronectin is a large glycoprotein (M_(r) ca 450 kd) with two similar subunits, which may vary in molecular size depending on a complex splicing pattern of a precursor mRNA (1). The major function of fibronectin, which is found in body fluids, blood clots and extracellular matrices, seems to be related to the ability of the protein to mediate substrate adhesion of most eukaryotic cells (2, 3, 4, 5.)

In the late seventies, Kuusela found that fibronectin not only interacts with eucaryotic cells but also binds to cells of Staphylococcus aureus (6). Since this observation, a number of pathogenic microorganisms have been shown to bind to fibronectin with a high degree of specificity and a high affinity, such as streptococci (group A, C, and G), coagulase negative staphylococci, E. coli and Treponema pallidum. Fibronectin in the extracellular matrix appears to serve as a substratum also for the adhesion of different microorganisms. The binding of fibronectin may for some microorganisms represent a crucial step in the colonization of host tissue and development of infection.

Several different cell surface components have been implicated as fibronectin receptors on Gram-positive bacteria including lipotechioc acid (8, 9) and protein (10). In previous studies a fibronectin binding protein with a M_(r) of 197-210 kD has been isolated from S. aureus strain Newman (11, 12) and tentatively identified as a fibronectin receptor. The binding site in fibronectin for eukaryotic cells has been localized to a tetrapeptide (ArgGlyAspSer) in the central portion of each of the two subunits forming the fibronectin, which is different to the binding site of most bacteria so far studied. The bacteria appear to bind to the aminoterminal 29 kDa domain of the fibronectin subunit.

An eukaryotic receptor has been identified as a 140 kDa complex in the cell membrane, whereas the bacterial fibronectin binding protein (FNBP) of Staphylococcus aureus strain Newman has been identified as a 210 kDa protein. From previous studies (SE-A-8702272-9) it has been reported of the cloning, expression and the complete nucleotide sequence of a gene (herein called gene 1) for a FNBP in Staphylococcus aureus.

In the present application the cloning, expression and the nucleotide sequence of a further gene, gene 2, located downstream the previous studied and reported fibronectin binding protein sequence. To further characterize this fibronectin binding protein from S aureus, the gene for this protein has been cloned in E. coli. The fibronectin binding domain within this protein has also been localized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1:1 to 1:3 depicts the nucleotide sequence of the nucleic acid encoding the fibronectin binding protein.

FIG. 2 presents a comparison between the amino acid sequences of the fibronectin binding proteins encoded by gene 1 and gene 2, respectively, which are given in parallel.

FIG. 3 is a restriction map of the original clones pFR0001 and pFR050 together with subclones pFR035 and pFR036. The location of fnbA and fnbB is indicated. The sequenced fragment of the insert is shown in more detail. The coding sequence in each clone are shown with bold lines.

FIG. 4 is the deduced amino acid sequence of the cloned fnbB from S. aureus strain 8325-4

FIG. 5 is a schematic drawing comparing domain organization of FnBPA and FnBPB.

DESCRIPTION OF THE INVENTION

It has now surprisingly been found possible to obtain a hybride-DNA-molecule comprising a nucleotide sequence coding for a protein or a polypeptide having fibronectin binding properties. As evident from below the following nucleotide sequence is present in the gene coding for said protein:

    __________________________________________________________________________     GTTAACAACA                                                                               ATCTTAACTT                                                                               TTTATTAACT                                                                               CGCTTTTTTT                                                                               CATTGCTTTT                             AAAAACCGAA                                                                               CAATATAGAA                                                                               TTGCATTTAT                                                                               TGAGTTTTTA                                                                               AAATAAATGA                             ATTTTGCATT                                                                               TAAGGGAGAA                                                                               TATTATAGTG                                                                               AAAAGCAATC                                                                               TTAGATACGG                             CATAAGAAAA                                                                               CACAAATTGG                                                                               GAGCGGCCTC                                                                               AGTATTCTTA                                                                               GGAACAATGA                             TCGTTGTTGG                                                                               AATGGGACAA                                                                               GAAAAAGAAG                                                                               CTGCAGCATC                                                                               GGAACAAAAC                             AATACTACAG                                                                               TAGAGGAAAG                                                                               TGGGAGTTCA                                                                               GCTACTGAAA                                                                               GTAAAGCAAG                             CGAAACACAA                                                                               ACAACTACAA                                                                               ATAACGTTAA                                                                               TACAATAGAT                                                                               GAAACACAAT                             CATACAGCGC                                                                               GACATCAACT                                                                               GAGCAACCAT                                                                               CACAATCAAC                                                                               ACAAGTAACA                             ACAGAAGAAG                                                                               CACCGAAAAC                                                                               TGTGCAAGCA                                                                               CCAAAAGTAG                                                                               AAACTTCGCG                             AGTTGATTTG                                                                               CCATCGGAAA                                                                               AAGTTGCTGA                                                                               TAAGGAAACT                                                                               ACAGGAACTC                             AAGTTGACAT                                                                               AGCTCAACAA                                                                               AGTAAAGTCT                                                                               CAGAAATTAA                                                                               ACCAAGAATG                             AAAAGATCAA                                                                               CTGACGTTAC                                                                               AGCAGTTGCA                                                                               GAGAAAGAAG                                                                               TAGTGGAAGA                             AACTAAAGCG                                                                               ACAGGTACAG                                                                               ATGTAACAAA                                                                               TAAAGTGGAA                                                                               GTAGAAGAAG                             GTAGTGAAAT                                                                               TGTAGGACAT                                                                               AAACAAGATA                                                                               CGAATGTTGT                                                                               AAATCCTCAT                             AACGCAGAAA                                                                               GAGTAACCTT                                                                               GAAATATAAA                                                                               TGGAAATTTG                                                                               GAGAAGGAAT                             TAAGGCGGGA                                                                               GATTATTTTG                                                                               ATTTCACATT                                                                               AAGCGATAAT                                                                               GTTGAAACTC                             ATGGTATCTC                                                                               AACACTGCGT                                                                               AAAGTTCCGG                                                                               AGATAAAAAG                                                                               TACAGATGGT                             CAAGTTATGG                                                                               CGACAGGAGA                                                                               AATAATTGGA                                                                               GAAAGAAAAG                                                                               TTAGATATAC                             GTTTAAAGAA                                                                               TATGTACAAG                                                                               AAAAGAAAGA                                                                               TTTAACTGCT                                                                               GAATTATCTT                             TAAATCTATT                                                                               TATTGATCCT                                                                               ACAACAGTGA                                                                               CGCAAAAAGG                                                                               TAACCAAAAT                             GTTGAAGTTA                                                                               AATTGGGTGA                                                                               GACTACGGTT                                                                               AGCAAAATAT                                                                               TTAATATTCA                             ATATTTAGGT                                                                               GGAGTTAGAG                                                                               ATAATTGGGG                                                                               AGTAACAGCT                                                                               AATGGTCGAA                             TTGATACTTT                                                                               AAATAAAGTA                                                                               GATGGGAAAT                                                                               TTAGTCATTT                                                                               TGCGTACATG                             AAACCTAACA                                                                               ACCAGTCGTT                                                                               AAGCTCTGTG                                                                               ACAGTAACTG                                                                               GTCAAGTAAC                             TAAAGGAAAT                                                                               AAACCAGGGG                                                                               TTAATAATCC                                                                               AACAGTTAAG                                                                               GTATATAAAC                             ACATTGGTTC                                                                               AGACGATTTA                                                                               GCTGAAAGCG                                                                               TATATGCAAA                                                                               GCTTGATGAT                             GTCAGCAAAT                                                                               TTGAAGATGT                                                                               GACTGATAAT                                                                               ATGAGTTTAG                                                                               ATTTTGATAC                             TAATGGTGGT                                                                               TATTCTTTAA                                                                               ACTTTAATAA                                                                               TTTAGACCAA                                                                               AGTAAAAATT                             ATGTAATAAA                                                                               ATATGAAGGG                                                                               TATTATGATT                                                                               CAAATGCTAG                                                                               CAACTTAGAA                             TTTCAAACAC                                                                               ACCTTTTTGG                                                                               ATATTATAAC                                                                               TATTATTATA                                                                               CAAGTAATTT                             AACTTGGAAA                                                                               AATGGCGTTG                                                                               CATTTTACTC                                                                               TAATAACGCT                                                                               CAAGGCGACG                             GCAAAGATAA                                                                               ACTAAAGGAA                                                                               CCTATTATAG                                                                               AACATAGTAC                                                                               TCCTATCGAA                             CTTGAATTTA                                                                               AATCAGAGCC                                                                               GCCAGTGGAG                                                                               AAGCATGAAT                                                                               TGACTGGTAC                             AATCGAAGAA                                                                               AGTAATGATT                                                                               CTAAGCCAAT                                                                               TGATTTTGAA                                                                               TATCATACAG                             CTGTTGAAGG                                                                               TGCAGAAGGT                                                                               CATGCAGAAG                                                                               GTACCATTGA                                                                               AACTGAAGAA                             GATTCTATTC                                                                               ATGTAGACTT                                                                               TGAAGAATCG                                                                               ACACATGAAA                                                                               ATTCAAAACA                             TCATGCTGAT                                                                               GTTGTTGAAT                                                                               ATGAAGAAGA                                                                               TACAAACCCA                                                                               GGTGGTGGTC                             AGGTTACTAC                                                                               TGAGTCTAAC                                                                               CTAGTTGAAT                                                                               TTGACGAAGA                                                                               TTCTACAAAA                             GGTATTGTAA                                                                               CTGGTGCTGT                                                                               TAGCGATCAT                                                                               ACAACAATTG                                                                               AAGATACGAA                             AGAATATACG                                                                               ACTGAAAGTA                                                                               ACTTGATTGA                                                                               ACTAGTAGAT                                                                               GAACTACCTG                             AAGAACATGG                                                                               TCAAGCGCAA                                                                               GGACCAATCG                                                                               AGGAAATTAC                                                                               TGAAAACAAT                             CATCATATTT                                                                               CTCATTCTGG                                                                               TTTAGGAACT                                                                               GAAAATGGTC                                                                               ACGGTAATTA                             TGGCGTGATT                                                                               GAAGAAATCG                                                                               AAGAAAATAG                                                                               CCACGTGGAT                                                                               ATTAAGAGTG                             AATTAGGTTA                                                                               CGAAGGTGGC                                                                               CAAAATAGCG                                                                               GTAATCAGTC                                                                               ATTTGAGGAA                             GACACAGAAG                                                                               AAGATAAACC                                                                               GAAATATGAA                                                                               CAAGGTGGCA                                                                               ATATCGTAGA                             TATCGATTTC                                                                               GATAGTGTAC                                                                               CTCAAATTCA                                                                               TGGTCAAAAT                                                                               AATGGTAACC                             AATCATTCGA                                                                               AGAAGATACA                                                                               GAGAAAGACA                                                                               AACCTAAGTA                                                                               TGAACAAGGT                             GGTAATATCA                                                                               TTGATATCGA                                                                               CTTCGACAGT                                                                               GTGCCACATA                                                                               TTCACGGATT                             CAATAAGCAC                                                                               ACTGAAATTA                                                                               TTGAAGAAGA                                                                               TACAAATAAA                                                                               GATAAACCAA                             ATTATCAATT                                                                               CGGTGGACAC                                                                               AATAGTGTTG                                                                               ACTTTGAAGA                                                                               AGATACACTT                             CCACAAGTAA                                                                               GTGGTCATAA                                                                               TGAAGGTCAA                                                                               CAAACGATTG                                                                               AAGAAGATAC                             AACACCTCCA                                                                               ATCGTGCCAC                                                                               CAACGCCACC                                                                               GACACCAGAA                                                                               GTACCAAGCG                             AGCCGGAAAC                                                                               ACCAACACCA                                                                               CCGACACCAG                                                                               AAGTACCAAG                                                                               CGAGCCGGAA                             ACACCAACAC                                                                               CGCCAACGCC                                                                               AGAGGTACCA                                                                               ACTGAACCTG                                                                               GTAAACCAAT                             ACCACCTGCT                                                                               AAAGAAGAAC                                                                               CTAAAAAACC                                                                               TTCTAAACCA                                                                               GTGGAACAAG                             GTAAAGTAGT                                                                               AACACCTGTT                                                                               ATTGAAATCA                                                                               ATGAAAAGGT                                                                               TAAAGCAGTG                             GTACCAACTA                                                                               AAAAAGCACA                                                                               ATCTAAGAAA                                                                               TCTGAACTAC                                                                               CTGAAACAGG                             TGGAGAAGAA                                                                               TCAACAAACA                                                                               ACGGCATGTT                                                                               GTTCGGCGGA                                                                               TTATTTAGCA                             TTTTAGGTTT                                                                               AGCGTTATTA                                                                               CGCAGAAATA                                                                               AAAAGAATCA                                                                               CAAAGCATAA                             TCAATCCAAA                                                                               ATTGACAGGT                                                                               TTATTTCATA                                                                               AATTATATGA                                                                               AGTAAGCCTG                             TTTTTTAAAA                                                                               TTAAAACAAA                                                                               TTTCCCAAGA                                                                               AATAATTACA                                                                               TACTCAATGA                             CACTATGAAG                                                                               GCGTTCTAAT                                                                               TAGTGTTAAA                                                                               ATGACGTTGA                                                                               TACATAGATT                             TAATACTTAG                                                                               GAAAAGGAGC                                                                               ACATTAACTT                                                                               TGAAAAAAAT                                                                               AAAAAAGGCA                             ATCATTCCCG                                                                               CTGCTGGTTT                                                                               AGGGACTAGA                                                                               TTTTTACCAG                                                                               CAACTAAAGC                             GATGCCAAAG                                                                               GAAATGCTTC                                                                               CTATCTTAGA                                                                               TAAACCCACA                                                                               ATACAATATA                             TCGTTGAAGA                                                                               AGCTGCAAGA                                                                               GCTGGAATTG                                                                               AAGATATTAT                                                                               TATAGTGACA                             GGTCGCCACA                                                                               AACGCGCGAT                                                                               TGAAGATCAT                                                                               TTTGATAGTC                                                                               AAAAAGAATT                             AGAAATGGTG                                                                               TTAAAAGAAA                                                                               AAGGTAAATC                                                                               TGAATTACTA                                                                               GAGAAAGTTC                             AGTATTCAAC                                                                               GGAACTTGCG                                                                               AATATTTTTT                                                                               ATGTAAGGCA                                                                               GAAAGAACAA                             AAAGGTTTAG                                                                               GGCATGC                                                              __________________________________________________________________________

whereby this nucleotide sequence encodes for the following protein starting at nucleotide no. 128 in the reading above, whereby the prepresent nucleotides are part of the signal system:

    __________________________________________________________________________     VKSNLRYGIR                                                                              KHKLGAASVF                                                                               LGTMIVVGMG                                                                              QEKEAAASEQ                                                                              NNTTVEESGS                                SATESKASET                                                                              QTTTNNVNTI                                                                               DETQSYSATS                                                                              TEQPSQSTQV                                                                              TTEEAPKTVO                                APKVETSRVD                                                                              LPSEKVADKE                                                                               TTGTQVDIAQ                                                                              QSKVSEIKPR                                                                              MKRSTDVTAV                                AEKEVVEETK                                                                              ATGTDVTNKV                                                                               EVEEGSEIVG                                                                              HKQDTNVVNP                                                                              HNAERVTLKY                                KWKFGEGIKA                                                                              GDYFDFTLSD                                                                               NVETHGISTL                                                                              RKVPEIKSTD                                                                              GQVMATGEII                                GERKVRYTFK                                                                              EYVQEKKDLT                                                                               AELSLNLFID                                                                              PTTVTQKGNQ                                                                              NVEVKLGETT                                VSKIFNIQYL                                                                              GGVRDNWGVT                                                                               ANGRIDTLNK                                                                              VDGKFSHFAY                                                                              MKPNNQSLSS                                VTVTGQVTKG                                                                              NKPGVNNPTV                                                                               KVYKHIGSDD                                                                              LAESVYAKLD                                                                              DVSKFEDVTD                                NMSLDFDTNG                                                                              GYSLNFNNLD                                                                               QSKNYVIKYE                                                                              GYYDSNASNL                                                                              EFQTHLFGYY                                NYYYTSNLTW                                                                              KNGVAFYSNN                                                                               AQGDGKDKLK                                                                              EPIIEHSTPI                                                                              ELEFKSEPPV                                EKHELTGTIE                                                                              ESNDSKPIDF                                                                               EYHTAVEGAE                                                                              GHAEGTIETE                                                                              EDSIHVDFEE                                STHENSKHHA                                                                              DVVEYEEDTN                                                                               PGGGQVTTES                                                                              NLVEFDEDST                                                                              KGIVTGAVSD                                HTTIEDTKEY                                                                              TTESNLIELV                                                                               DELPEEHGQA                                                                              QGPIEEITEN                                                                              NHHISHSGLG                                TENGHGNYGV                                                                              IEEIEENSHV                                                                               DIKSELGYEG                                                                              GQNSGNQSFE                                                                              EDTEEDKPKY                                EQGGNIVDID                                                                              FDSVPQIHGQ                                                                               NNGNQSFEED                                                                              TEKDKPKYEQ                                                                              GGNIIDIDFD                                SVPHIHGFNK                                                                              HTEIIEEDTN                                                                               KDKPNYQFGG                                                                              HNSVDFEEDT                                                                              LPQVSGHNEG                                QQTIEEDTTP                                                                              PIVPPTPPTP                                                                               EVPSEPETPT                                                                              PPTPEVPSEP                                                                              ETPTPPTPEV                                PTEPGKPIPP                                                                              AKEEPKKPSK                                                                               PVEQGKVVTP                                                                              VIEINEKVKA                                                                              VVPTKKAQSK                                KSELPETGGE                                                                              ESTNNGMLFG                                                                               GLFSILGLAL                                                                              LRRNKKNHKA                                         __________________________________________________________________________

In the single letter amino acid sequence above the following abbreviations have been used

A Ala, Alanine

R Arg, Arginine

N Asn, Asparagine

D Asp, Aspartic acid

C Cys, Cysteine

C Cys, Cysteine

G Gly, Clycine

E Glu, Glutamic acid

Q Gln, Glutamine

H His, Histidine

I Ile, Isoleucine

L Leu, Leucine

K Lys, Lysine

M Met, Methionine

F Phe, Phenylalanine

P Pro, Proline

S Ser, Serine

T Thr, Threonine

W Trp, Tryptophan

Y Tyr, Tyrosine

V Val, Valine

Above, the nucleotide sequence of the starting signal ends at nucleotide 235 and the sequence starting at nucleotide no. 1735 shows the nucleotide sequence of the binding region, which corresponds to the following amino acid sequence

    __________________________________________________________________________     IETEEDSIHV                                                                              DFEESTHHEN                                                                              SKHHADVVEY                                                                              EEDTNPGGGQ                                                                              VTTESNLVEF                                 DEDSTKGIVT                                                                              GAVSDHTTIE                                                                              DTKEYTTESN                                                                              LIELVDELPE                                                                              EHGQAQGPIE                                 EITENNHHIS                                                                              HSGLGTENGH                                                                              GNYGVIEEIE                                                                              ENSHVDIKSE                                                                              LGYEGGQNSG                                 NQSFEEDTEE                                                                              DKPKYEQGGG                                                                              NIVDIDFDSV                                                                              PQIHGQNNGN                                                                              QSFEEDTEKD                                 KPKYEQGGNI                                                                              IDIDFDSVPH                                                                              IHGFNKHTEI                                                                              IEEDTNKDKP                                                                              NYQFGGHNSV                                 DFEEDTLPQV                                                                              SGHNEGQQTI                                                                              EEDTTPPIVP                                                                              PTPPTPEVPS                                                                              EPETPTPPTP                                 EVPSEPETPT                                                                              PPTPEVPTEP                                                                              GKPIPPAKEE                                                                              PKKPSKPVEQ                                                                              GKVVTPVIEI                                 NEKVKAVVPT                                                                              KKAQSKKSEL                                                                              PETGGEESTN                                                                              NGMLFGGLFS                                                                              ILGLALLRRN                                                                              KKNHKA                            __________________________________________________________________________

The invention further comprises a plasmid or phage comprising a nucleotide sequence coding for said fibronectin binding protein.

The invention further comprises a microorganism containing at least one hybrid-DNA-molecule according to the above. The plasmid pFR001 in an E. coli strain 259 has been deposited at the Deutsche Sammlung von Mikroorganismen (DSM), and has thereby been allocated the deposition number DSM 4124.

The invention further comprises a method for producing a fibronectin binding protein whereby at least one hybrid-DNA-molecule of above is transferred into a microorganism, cultivating said microorganism in a growth medium, and isolating the protein thus formed by means of affinity chromatography on a coloumn containing fibronectin bound to an insolubilized carrier followed by ion exchange chromatography.

A further aspect of the invention comprises a chemical synthesis of the fibronectin binding protein, whereby an amino acid sequence is built up based on said nucleotide sequence encoding for said protein starting from the C-terminal alanine which is stepwise reacted with the appropriate amino acid, whereby it is finally reacted with isoleucine at the N-terminal end, to form the fibronectin binding peptide region.

Appropriate carrier proteins can be coupled to the amino acid sequence as well, such as IgG binding regions of protein A.

The invention will be described in the following with reference to the examples given, however, without being restricted thereto.

EXAMPLE

Chemical synthesis of a polypeptide based on the nucleotide sequence coding for the fibronectin binding domain was performed by building up the amino acid sequence corresponding to said nucleotide sequence starting from the C-terminal alanine and stepwise reacting with the appropriate amino acid and finally reacting with the isoleucine at the N-terminal end, in a solid phase synthesis according to the method by K. B. Merrifield, J. Am. Chem. Soc. 86, pp.304, (1964).

MATERIALS AND METHODS

Microorganism growth medium. For growth of E. coli bacteria the following medium was used. The amounts given relates to 1 litre of medium.

    ______________________________________                                         Trypton Soy Broth (Oxoid Ltd,                                                                          30     g                                               Basingstoke, Hants, GB)                                                        Yeast Extract (Oxoid)   10     g                                               D-glucose               40     g                                               NH.sub.4 Cl             2.5    g                                               Na.sub.2 HPO.sub.4.2H.sub.2 O                                                                          7.5    g                                               KH.sub.2 PO.sub.4       3.0    g                                               Na.sub.2 SO.sub.4.10H.sub.2 O                                                                          2.5    g                                               MgSO.sub.4.7H.sub.2 O   0.2    g                                               CaCl.sub.2.2H.sub.2 O   0.5    mg                                              FeCl.sub.3.6H.sub.2 O   16.7   mg                                              ZnSO.sub.4.7H.sub.2 O   0.18   mg                                              CuSO.sub.4.5H.sub.2 O   0.16   mg                                              MnSO.sub.4.4H.sub.2 O   0.15   mg                                              CoCl.sub.2              0.10   mg                                              NaEDTA                  20.1   mg                                              ______________________________________                                    

Assay of Fibronectin Binding Protein (FNBP)

Lysates of E. coli clones prepared in Tris-HCl buffer, containing lysozyme EDTA as earlier described (13), were analysed for fibronectin binding activity by measuring their ability to compete with staphylococcal cells for binding the ¹²⁵ I-labelled 29 kD NH₂ -terminal fragment of fibronectin. The amount of FNBP able to inhibit binding to 50% is considered as one unit of activity. Bovine fibronectin was provided by Dr. S. Johansson the Department of Medical and Physiological Chemistry, University of Uppsala, Sweden. Overnight cultures of E. coli were concentrated 10 times followed by lysis in 0.01M Tris-HCl, 0.001 EDTA, pH 7.9, 1 mg/ml of lysozyme. 100 μl lysate was mixed with 100 μl staphylococcal cells, 100 μl ¹²⁵ I bovinefibronectin (20000 cpm/ml), 200 μl PBS, and the mixture was incubated for 2 hrs at 20° C. After washing twice in PBS containing 0.1% BSA and 0.05% Tween the radioactivity of the mixture was measured in a gamma counter.

Iodinnation

¹²⁵ I-labelling of fibronectin and fibronectin fragments was performed using the chloramine-T method.

Bacterial Strains and Plasmids

E. coli TG-1 and DH-5alfa were used as bacterial hosts. The plasmid vectors were pBR322 and pUC18. Table 1 lists the plasmids.

Media and Growth Conditions

E. coli clones were grown in Luria Broth (LB) supplemented with ampicillin at 50 /μg/ml and shaken at 37° C. The optical density was measured with a Linson 3,1 Photometer read at 540 nm. S. aureus was grown in Trypticase Soya Broth (TSB).

Restriction endonucleases and other enzymes. Restriction enzymes, T4 DNA ligase and Bal31 were purchased from Promega (Madison, Wis.), International Biotechnologies Inc. (New Haven, Conn.) and Boehringer Mannheim Biochemicals Scandinavia AB. Restriction mapping and fragment isolation were performed with LiCl₄ extracted plasmid DNA. Cloning in pUC18 was performed as described by Maniatis et al. Generation of subclones for sequencing was performed by ExoIII digestion using Erase-a-Base System purchased from Promega. E. coli clones were verified by restriction analysis, sequence analysis, and blot hybridazation. DNA sequencing was done by the dideoxynucleotide methods of Sanger et al, with the sequenase DNA sequencing kit purchased from United States Biochemical Corporation Cleveland Ohio, and the K/RT universal sequencing system purchased from Promega. The sequencing samples were analysed by wedge shaped gels using 6% polyacrylamide. Computer programms were used to record and analyse the sequence data.

The isolation of an E. coli clone containing gene 1 and part of gene 2 for a FNBP from S. aureus strain 8325-4 was described earlier. The plasmid pFR050 was constructed from S. aureus by cleaving 8325-4 chromosomal DNA with HindIII and XbaI. Fragments, 3-4 kbp in size were isolated after agarose-gel electrofores and ligated into pUC18. One clone containing fnbB sequences was isolated by colony hybridization using a synthetic oligonucleotide located downstream the HindIII-site in fnbB as a probe. The oligonucleotide was synthetized with Applied Biosystem 380A oligonucleotide synthetizer using the phosphoamidite method. Computer programms were used to record and analyse the sequence data.

Western Blotting

Separated components were electroblotted onto NC-sheets (nitrocellulose sheets) (Schleicher and Schnell) for 2 hrs, 200V using the miniblot system (LKB) and the buffer system described by Towbin. Subsequently NC-sheets were saturated with 1% BSA in TBS, pH 7.4, for 30 min, and incubated with 2.4 μg/ml bovine fibronectin in TBS, pH 7.4, for 2 hrs. After washing three times using PBS-Tween (0.1%), the NC-sheets were incubated with rabbit anti bovine fibronectin serum diluted 1:1000, which serum was a gift from Biochemical Centre, University of Uppsala, for 1.5 hrs, followed by washing and final incubation with a protein A peroxidase conjugate (prepared from S aureus A676 protein by conjugation with horse radish peroxidase (Boehringer) in a molar ratio of 1:2) for 1.5 hrs. After final washings 3 times with PBS-Tween, 1x with PBS, the blot was developed with 4-chloro-1-naphtol (Sigma).

Cloning of a Gene Coding for a Second Fibronectin Binding Protein

In our previous work it was described the cloning, expression and determination of the sequence of a gene coding for a fibronectin binding protein (gene 1). In a further analysis of these older sequence data it was found a region, located downstream of gene 1, which showed high homology with the beginning of gene 1. In order to determine if this region downstream of gene 1 exhibits a fibronectin binding activity, a 2.8 kb PstI fragment from pFR001 containing a sequence starting 680 bp downstream the stopcodon of gene 1 was introduced into the multilinker of pUC18. Knowing the transcription direction of gene 2 and its reading frame (from left to right in FIG. 1) it was possible to fuse the fragment in the correct reading frame to the lac-Z promoter of pUC18. This plasmid called pFR035, expressed fibronectin binding activity (Table 1 below). Thus there exist two different genes encoding FnBPs. However, when sequencing pFR035 it could not be found any stop codon in the inserted S. aureus DNA, and by comparing fnbA (gene 1) it was obvious that the complete fnbB was not present. By making southern blots of chromosomal DNA cleaved with HindIII alone, and together with other enzymes, we found that digestion with HindIII together with XbaI would generate a 3.5 kbp fragment (including 65 bp already present in pFR035), which most likely also would contain the missing 3'-part of fnbB. The fragment was cloned as described above and was called pFR050.Subclones of the plasmid were derived by digestion of pFR035 with ExoIII from the 3' end for different time periods with subsequent religation of the DNA, as described in Materials and Methods, above.

TABLE 1

Origin and expression of fibronectin binding activity for clones discussed in this invention. Assay for fibronectin binding is described in Materials and Methods, above.

    ______________________________________                                         Clone   Derivation             Fn-binding                                      ______________________________________                                         pFR001  Original isolate       +                                               pFR035  2.8 kb PstI fragment from pFR001                                                                      +                                               pFR036  2.3 kb HpaI/EcoRI fragment                                                                            +                                                       from pFR001                                                            pFR035e31                                                                              pFR035 with 1.3 kb deleted from the 3'                                                                -                                                       PstI site (of which 1.1 kb is                                                  vector DNA)                                                            pFR035e35                                                                              as pFR035e31 but 1.47 kb deleted                                                                      -                                               pFR050  Original isolate       +                                               pFR060  2.0 kbp NheI/SphI fragment from                                                                       +                                                       pFR050 inserted into pFR035 opened                                             with NheI/SphI                                                         ______________________________________                                    

Sequence Analysis

A nucleotide sequence of 1928 bp containing a domain encoding a fibronectin binding protein was determined by sequencing the overlapping subclones derived from pFR035 and pFR001 (FIG. 2). One open reading frame encodes a polypeptide of 940 amino acids, starting with a GTG codon at nucleotide 520, and terminating at the end of the clone at nucleotide 3342 (FIG. 2). FnbB, as fnbA (gene 1) has two possible initiation signals for transcription and a potential ribosome binding site (marked in FIG. 2). The start codon is followed by a possible signal sequence which shows 95% homology to that encoded by fnbA (FIG. 2, and 4). By comparison to FnBPA the cleavage site of the signal sequence is located between the second and third in row of three alanine residues. This corresponds to the cleavage site for the native protein isolated from S. aureus strain Newman. Downstream the signal sequence there is a stretch of about 66 amino acids with a 75% homology to the same stretch in fnbA. The following 444 amino acids have only 40% homology towards FnBPA and have several deletions/insertions, so the B-repeats found in FnBPA is not seen in FnBPB (FIG. 2 and 4). However the rest of the peptide (394 aa) is nearly identical to FnBPA, the main difference being the deletion of 14 amino acids in FnBPB. This highly homologous region contains the same repeat (D1-D4 and Wr1-5) found in FnBPA with the exception that Wr1 is lacking. The Wc region and the hydrophobic region M domain as well as the mainly basic C-terminal end is conserved in FnBPB.

Expression of Fibronectin Binding Protein and Identification of the Binding Activity

The E. coli clones pFR035 and pFR036 and subclones derived by deleting the gene 2 fragment of pFR035 were lysed and tested for fibronectin binding protein activity in the inhibition assay. Lysate of both clones inhibit ¹²⁵ I-labelled fibronectin to bind to S. aureus, whereas the subclone pFR035e31, deleted from the 3' terminal of the gene 2 fragment, has lost the activity (FIG. 3). The fibronectin binding protein activity is thus located to the amino acids downstream amino acid no. 535 (FIG. 1). None of these clones include the D-repeates which has been shown to be the only Fn-binding domain in FnBPA. This will imply that FnBPB contains two different Fn-binding domains one region upstream of amino acid 600 and the D-region. Assay of the FnBp. E. coli clones containing different parts of the fnbB were analysed for Fn-binding activity by measuring their ability to compete with staphylococcal cells for binding of ¹²⁵ I-labelled intact bovine Fn or the 29 kDa N-terminal fragment. Over night cultures of E. coli were concentrated 10 times and lysed in 10 mM Tris-HCl, 1 mM EDTA, pH 7.9, 1 mg/ml lysozyme. 100 /μl supernatant of centrifuged lysate was mixed with 100 /μl staphylococcal cells (5×10⁸), 100 μl ¹²⁵ I-bovine Fn (20,000 cpm, 190 MBq/mg), 200 μl PBS and incubated 2 hrs at 20° C. After washing the mixture twice in PBS containing 0.1% BSA and 0.05 Tween^(R) 20, the radioactivity bound to the bacterial cells was measured in a gamma counter.

Iodination, ¹²⁵ I-labelling of Fn and Fn fragments were done according to the chloramine-T method.

Molecular Weight Determination

Western blotting of lysate from pFR035 shows a band corresponding to a molecular weight of 100 kDa and several bands of lower molecular weight, which most likely are degradation products of the 100 kDa product since a shift to lower molecular weights is seen upon storage of the material. The difference seen in the processing is probably due to the fact that in pFR035 the FnBPB is fused to the beta-Gal protein, but in pFR036 it utilizes its own initiation signals, so the proteins are slightly different.

The data presented demonstrate that S. aureus has two different genes encoding for FnBPs. The start codon of fnbB is situated 682 bp downstream the stop codon of fnbA. This sequence between fnbA and fnbB contains a possible transcription termination signal located just a few bp downstream the said stop codon. as well as transcription initiation signals located within the 90 bps which preceeds the start codon in fnbB. This implies that the genes are translated from different messenger RNAs. The region between these transcriptional signals does not contain any open reading frames preceeded by a ribosomal binding site on either strand. The 350 bp region upstream the promotor sequence of fnbB show strong homology with the analogous region of fnbA. In fnbA the binding activity has been localised to the D-repeate domain (between aa 745 and 872) near the cell wall associated part of the molecule, and a subclone where amino acids 746-1018 was excluded was Fn-binding negative. When the two genes are compared it is evident that there is no repeat region present in the pFR035 and pFR036. Still both express Fn-binding activity, which indicates that a non-homologous nucleotide sequence is present encoding for Fn-binding activity.

The expression of the fibronectin binding protein from gene 2 in E. coli, was lower than expression of gene 1.

The present fibronectin binding protein can be used for immunization, whereby the protein, preferably in combination with a fusion protein to create a large antigen to respond to, is injected in dosages causing immunological reaction in the host mammal. Thus the fibronectin binding protein can be used in vaccination of ruminants against mastitis caused by Staphylococcal infections.

Further, the fibronectin binding protein can be used to block an infection in an open skin wound by wound treatment using the fibronectin binding protein in a suspension. Thus the fibronectin binding protein can be used for the treatment of wounds, e.g. for blocking protein receptors, or for immunization (vaccination). In the latter case the host body produces specific antibodies, which can protect against invasion of bacterial strains comprising such a fibronectin binding protein. Hereby the antibodies block the adherence of the bacterial strains to damaged tissue. Examples of colonizing of a tissue damage are:

a) colonizing of wounds in skin and connective tissue, which wounds have been caused by a mechanical trauma, chemical damage, and/or thermical damage;

b) colonizing of wounds on mucous membranes, such as in the mouth cavity, or in the mammary glands, urethra, or vagina;

c) colonizing on connective tissue proteins, which have been exposed by a minimal tissue damage (microlesion) in connection with epithelium and endothelium (mastitis, heart valve infection, hip exchange surgery).

When using the present FNBP, or the polypeptide, for the purpose of immunization (vaccination) in mammals, including man, the protein, or polypeptide is dispersed in sterile, isotonic saline solution, optionally while adding a pharmaceutically acceptable dispersing agent. Different types of adjuvants can further be used in order to sustain the release in the tissue, and thus expose the protein or the peptide for a longer time to the immundefense system of a body.

A suitable dosage to obtain immunization is 0,5 to 5 μg of FNBP, or polypeptide, per kg bodyweight and injection of immunization. In order to obtain a durable immunization, vaccination should be carried out at more than one consecutive occasions with an interval of 1 to 3 weeks, preferably at three occasions.

When using the present FNBP, or polypeptide, for topical, local administration the protein is dispersed in an isotonic saline solution to a concentration of 25 to 250 μg per ml. The wounds are then treated with such an amount only to obtain a complete wetting of the wound surface. For an average wound thus only a couple of millilitres of solution are used in this way. After treatment using the protein solution the wounds are suitably washed with isotonic saline or another suitable wound treatment solution.

Further the fibronectin binding protein as well as the minimal fibronectin binding site polypeptide, of the present invention can be used to diagnose bacterial infections caused by Staphylococci strains, whereby a fibronectin binding protein of the present invention is immobilized on a solid carrier, such as small latex or Sepharose^(R) beads, whereupon sera containing antibodies are allowed to pass and react with the FNBP thus immobilized. The agglutination is then measured by known methods.

Further, the FNBP, or the polypeptide can be used in an ELISA test (Enzyme Linked Immuno Sorbet Assay; E Engvall, Med. Biol. 55, 193, (1977)). Hereby wells in a polystyrene microtitre plate are coated with the FNBP, and incubated over night at 4° C. The plates are then thoroughly washed using PBS containing 0.05% TWEEN 20, and dried. Serial dilution of the patient serum were made in PBS-Tween, were added to the wells, and incubated at 30° C. for 1.5 hrs. After rinsing antihuman-IgG conjugated with an enzyme, or an antibovine-IgG conjugated with an enzyme, respectively, horseradishperoxidase or an alkaline phosphatase, was added to the wells and incubated at 30° C. for 1,5 hrs, whereupon when the IgG has been bound thereto, and after rinsing, an enzyme substrate is added, a p-nitrophosphate in case of an alkaline phosphatase, or ortophenylene diamine substrate (OPD) in case a peroxidase has been used, respectively. The plates comprising the wells were thus then rinsed using a citrate buffer containing 0.055% OPD, and 0.005% H₂ O₂, and incubated at 30° C. for 10 min. Enzyme reaction was stopped by adding a 4N solution of H₂ SO₄ to each well. The colour development was measured using a spectrophotometer.

Depending on the type of enzyme substrate used a fluoroscense measurement can be used as well.

Another method to diagnose Staphylococci infections is by using the DNA gene probe method based on the FNBP sequence or the polypeptide sequence. Thereby the natural or synthetic DNA sequences are attached to a solid carrier, such as a polystyrene plate as mentioned above, by e.g. adding a milk in the case of diagnozing a mastitis, to the surface. The DNA gene probe, optionally labelled enzymatically, or by a radioactive isotope is then added to the solid surface plate comprising the DNA sequence, whereby the DNA gene probe attaches to the sequence where appearing. The enzyme or the radioactive isotope can then readily be determined by known methods.

Above the term fibronectin binding protein includes the polypeptide sequence as well, which polypeptide sequence forms the minimal fibronectin binding site of the complete protein.

REFERENCES

8. Beachey, E. H. and Simpson, W. A. (1982). Infection 10, 107-110.

9. Courtney, H. S., Ofek, I., Simpson, W. A. Hasty, D. L. and Beachey, E. H. (1986). Infect. Immun. 53, 454-459.

11. Espersen, F. and Clemmensen, I. (1982). Infect. Immun. 37, 526-531.

12. Froman, G., Switalski, L. M., Speziale, P. and Hook, M. (1987). J. Biol. Chem. 262, 2564-2571

1. Hynes, R. O. (1985) Annu. Rev. Cell Biol. 1, 67-90.

2. Hynes, R. O. (1986) Sci. Ann. 254, 42-51.

6. Kuusela, P. (1978) Nature 276, 718-720.

13. Lofdahl, S., Guss B., Uhlen, M., Philipson, L. and Lindberg, M. (1983) Proc. Natl. Acad. Sci. USA 80, 697-701.

3. Ruoslahti, E. and Pierschbacher, M. D. (1986). Cell, 44, 517-518.

10. Ryden, C., Rubin, K., Speziale, P., Hook, M., Lindberg, M. and Wadstrom, T. (1983), J. Biol. Chem. 258, 3396-3401.

4. Woods, A., Couchman, J. R., Johansson, S., and Hook, M. (1986), EMBO J. 5, 665-670.

5. Yamada, K. M. (1983), Annu. Rev. Biochem. 52, 761-799. 

We claim:
 1. A hybrid DNA molecule comprising a nucleotide sequence from S. aureus coding for a protein or polypeptide having fibronectin binding activity, said nucleotide sequence comprising:

    ______________________________________                                         GTTAACAACA  ATCTTAACTT     TTTATTAACT                                          AAAAACCGAA  CAATATAGAA     TTGCATTTAT                                          ATTTTGCATT  TAAGGGAGAA     TATTATAGTG                                          CATAAGAAAA  CACAAATTGG     GAGCGGCCTC                                          TCGTTGTTGG  AATGGGACAA     GAAAAAGAAG                                          AATACTACAG  TAGAGGAAAG     TGGGAGTTCA                                          CGAAACACAA  ACAACTACAA     ATAACGTTAA                                          CATACAGCGC  GACATCAACT     GA GCAACCAT                                         ACAGAAGAAG  CACCGAAAAC     TGTGCAAGCA                                          AGTTGATTTG  CCATCGGAAA     AAGTTGCTGA                                          AAGTTGACAT  AGCTCAACAA     AGTAAAGTCT                                          AAAAGATCAA  CTGACGTTAC     AGCAGTTGCA                                          AACTAAAGCG  ACAGGTACAG     ATGTAACAAA                                          GTAGTGAAAT  TGTAGGACAT     AAACAAGATA                                          AACGCAGAAA  GAGTAACCTT     GAAATATAAA                                          TAAGGCGGGA  GATTATTTTG     ATTTCACAT T                                         ATGGTATCTC  AACACTGCGT     AAAGTTCCGG                                          CAAGTTATGG  CGACAGGAGA     AATAATTGGA                                          GTTTAAAGAA  TATGTACAAG     AAAAGAAAGA                                          TAAATCTATT  TATTGATCCT     ACAACAGTGA                                          GTTGAAGTTA  AATTGGGTGA     GACTACGGTT                                          ATATTTAGGT  GGAGTTAGAG     ATAATTGGGG                                          TTGATACTTT  AAATAAAGTA     GATGGGAAAT                                          AAACCTAACA  ACCAGTCGTT     AAGCTCTGTG                                          TAAAGGAAAT  AA ACCAGGGG    TTAATAATCC                                          ACATTGGTTC  AGACGATTTA     GCTGAAAGCG                                          GTCAGCAAAT  TTGAAGATGT     GACTGATAAT                                          TATTGGTGGT  TATTCTTTAA     ACTTTAATAA                                          ATGTAATAAA  ATATGAAGGG     TATTATGATT                                          TTTCAAACAC  ACCTTTTTGG     ATATTATAAC                                          AACTTGGAAA  AATGGCGTTG     CATTTTACTC                                          GCAAAGATAA  ACTAAAGGAA     CCTATTATAG                                          CTTGAATT TA AATCAGAGCC     GCCAGTGGAG                                          AATCGAAGAA  AGTAATGATT     CTAAGCCAAT                                          CTGTTGAAGG  TGCAGAAGGT     CATGCAGAAG                                          GA TTCTATTC ATGTAGACTT     TGAAGAATCG                                          TCATGCTGAT  GTTGTTGAAT     ATGAAGAAGA                                          AGGTTACTAC  TGAGTCTAAC     CTAGTTGAAT                                          GGTATTGTAA  CTGGTGCTGT     TAGCGATCAT                                          AGAATATACG  ACTGAAAGTA     ACTTGATTGA                                          AAGAACATGG  TCAAGCGCAA     GGACCA ATCG                                         CATCATATTT  CTCATTCTGG     TTTAGGAACT                                          TGGCGTGATT  GAAGAAATCG     AAGAAAATAG                                          AATTAGGTTA  CGAAGGTGGC     CAAAATAGCG                                          GACACAGAAG  AAGATAAACC     GAAATATGAA                                          TATCGATTTC  GATAGTGTAC     CTCAAATTCA                                          AATCATTCGA  AGAAGATACA     GAGAAAGACA                                          GGTAATATCA  TTGATATCGA     CTTCGACAGT                                          CAATAAGCAC  ACTGAAATTA     TTGAAGAAGA                                          ATTATCAATT  CGGTGGACAC     AATAGTGTTG                                          CCACAAGTAA  GTGGTCATAA     TGAAGGTCAA                                          AACACCTCCA  ATCGTGCCAC     CAACGCCACC                                          AGCCGGAAAC  ACCAACACCA     CCGACACCAG                                          ACACCAACAC  CGCCAACGCC     AGAGGTACCA                                          ACCACCTGCT  AAAGAAGAAC     CTAAAAAACC                                          GTAAAGTAGT  AACACCTGTT     ATTGAAATCA                                          GTACCAACTA  AAAAAGCACA     ATCTA AGAAA                                         TGGAGAAGAA  TCAACAAACA     ACGGCATGTT                                          TTTTAGGTTT  AGCGTTATTA     CGCAGAAATA                                          TCAATCCAAA  ATTGACAGGT     TTATTTCATA                                          TTTTTTAAAA  TTAAAACAAA     TTTCCCAAGA                                          CACTATGAAG  GCGTTCTAAT     TAGTGTTAAA                                          TAATACTTAG  GAAAAGGAGC     ACATTAACTT                                          ATCATTCCCG  CTGCTGGTTT     AGGGACTAGA                                                   CGCTTTTTTT   CATTGCTT TT                                                       TGAGTTTTTA   AAATAAATGA                                                        AAAAGCAATC   TTAGATACGG                                                        AGTATTCTTA   GGAACAATGA                                                        CTGCAGCATC   GGAACAAAAC                                                        GCTACTGAAA   GTAAAGCAAG                                                        TACAATAGAT   GAAACACAAT                                                        CACAATCAAC   ACAAGTAACA                                                        CCAAAAGTAG   AAACTTCGCG                                                        TAAGGAAACT   ACAGGAACTC                                                        CAGAAATTAA   ACCAAGAATG                                                        GAGAAAGAAG   TAGTGGAAGA                                                        TAAAGTGGAA   GTAGAAGAAG                                                        CGAATGTTGT   AAATCCTCAT                                                        TGGAAATTTG   GAGAAGGAAT                                                        AAGCGATAAT   GTTGAAACTC                                                        AGATAAAAAG   TACAGATGGT                                                        GAAAGAAAAG   TTAGATATAC                                                        TTTAACTGCT   GAATTATCTT                                                        CGCAAAAAGG   TAACCAAAAT                                                        AGCAAAATAT   TTAATATTCA                                                        AGTAACAGCT   AATGGTCGAA                                                        TTAGTCATTT   TGCGTACATG                                                        ACAGTAACTG   GTCAAGTAAC                                                        AACAGTTAAG   GTATATAA AC                                                       TATATGCAAA   GCTTGATGAT                                                        ATGAGTTTAG   ATTTTGATAC                                                        TTTAGACCAA   AGTAAAAATT                                                        CAAATGCTAG   CAACTTAGAA                                                        TATTATTATA   CAAGTAATTT                                                        TAATAACGCT   CAAGGCGACG                                                        AACATAGTAC   TCCTATCGAA                                                        AAGCATGAAT   TGACTGGTAC                                                        TGATTTTGAA   TATCATACAG                                                        GTACCATTGA   AACTGAAGAA                                                        ACACATGAAA   ATTCAAAACA                                                        TACAAAC CCA  GGTGGTGGTC                                                        TTGACGAAGA   TTCTACAAAA                                                        ACAACAATTG   AAGATACGAA                                                        ACTAGTAGAT   GAACTACCTG                                                        AGGAAATTAC   TGAAAACAAT                                                        GAAAATGGTC   ACGGTAATTA                                                        CCACGTGGAT   ATTAAGAGTG                                                        GTAATCAGTC   ATTTGAGGAA                                                        CAAGGTGGCA   ATATCGTAGA                                                        TGGTCAAAAT   AATGGTAACC                                                        AACCTAAGTA   TGAACAAGGT                                                        GTGCCACATA   TTCACGGATT                                                        TACAAATAAA   GATAAACC AA                                                       ACTTTGAAGA   AGATACACTT                                                        CAAACGATTG   AAGAAGATAC                                                        GACACCAGAA   GTACCAAGCG                                                        AAGTACCAAG   CGAGCCGGAA                                                        ACTGAACCTG   GTAAACCAAT                                                        TTCTAAACCA   GTGGAACAAG                                                        ATGAAAAGGT   TAAAGCAGTG                                                        TCTGAACTAC   CTGAAACAGG                                                        GTTCGGCGGA   TTATTTAGCA                                                        AAAAGAATCA   CAAAGCATAA                                                        AATTATATGA   AGTAAGCCTG                                                        AATAATTACA   TACTCAA TGA                                                       ATGACGTTGA   TACATAGATT                                                        TGAAAAAAAT   AAAAAAGGCA                                                        TTTTTACCAG   CAACTAAAGC.                                              ______________________________________                                    


2. A microorganism transformed by the hybrid DNA molecule of claim
 1. 3. A method for producing a fibronectin binding protein or polypeptide, wherein a) at least one hybrid-DNA-molecule according to claim 1 is introduced into a microorganism, b) said microorganism is cultivated in a growth promoting medium, and c) the protein thus formed is isolated by means of an affinity chromotography on a coloumn with fibronectin bound to an insolubilized carrier followed by ion exchange chromatography.
 4. A plasmid or phage comprising a nucleotide sequence from S. aureus coding for a protein or polypeptide having fibronectin binding activity, said nucleotide sequence comprising:

    __________________________________________________________________________     GTTAACAACA                                                                               ATCTTAACTT                                                                               TTTATTAACT                                                                               CGCTTTTTTT                                                                               CATTGCTTTT                             AAAAACCGAA                                                                               CAATATAGAA                                                                               TTGCATTTAT                                                                               TGAGTTTTTA                                                                               AAATAAATGA                             ATTTTGCATT                                                                               TAAGGGAGAA                                                                               TATTATAGTG                                                                               AAAAGCAATC                                                                               TTAGATACGG                             CATAAGAAAA                                                                               CACAAATTGG                                                                               GAGCGGCCTC                                                                               AGTATTCTTA                                                                               GGAACAATGA                             TCGTTGTTGG                                                                               AATGGGACAA                                                                               GAAAAAGAAG                                                                               CTGCAGCATC                                                                               GGAACAAAAC                             AATACTACAG                                                                               TAGAGGAAAG                                                                               TGGGAGTTCA                                                                               GCTACTGAAA                                                                               GTAAAGCAAG                             CGAAACACAA                                                                               ACAACTACAA                                                                               ATAACGTTAA                                                                               TACAATAGAT                                                                               CAAACACAAT                             CATACAGCGC                                                                               GACATCAACT                                                                               GAGCAACCAT                                                                               CACAATCAAC                                                                               ACAAGTAACA                             ACAGAAGAAG                                                                               CACCGAAAAC                                                                               TGTGCAAGCA                                                                               CCAAAAGTAG                                                                               AAACTTCGCG                             AGTTGATTTG                                                                               CCATCGGAAA                                                                               AAGTTGCTGA                                                                               TAAGGAAACT                                                                               ACAGGAACTC                             AAGTTGACAT                                                                               AGCTCAACAA                                                                               AGTAAAGTCT                                                                               CAGAAATTAA                                                                               ACCAAGAATG                             AAAAGATCAA                                                                               CTGACGTTAC                                                                               AGCAGTTGCA                                                                               GAGAAAGAAG                                                                               TAGTGGAAGA                             AACTAAAGCG                                                                               ACAGGTACAG                                                                               ATGTAACAAA                                                                               TAAAGTGGAA                                                                               GTAGAAGAAG                             GTAGTGAAAT                                                                               TGTAGGACAT                                                                               AAACAAGATA                                                                               CGAATGTTGT                                                                               AAATCCTCAT                             AACGCAGAAA                                                                               GAGTAACCTT                                                                               GAAATATAAA                                                                               TGGAAATTTG                                                                               GAGAAGGAAT                             TAAGGCGGGA                                                                               GATTATTTTG                                                                               ATTTCACATT                                                                               AAGCGATAAT                                                                               GTTGAAACTC                             ATGGTATCTC                                                                               AACACTGCGT                                                                               AAAGTTCCGG                                                                               AGATAAAAAG                                                                               TACAGATGGT                             CAAGTTATGG                                                                               CGACAGGAGA                                                                               AATAATTGGA                                                                               GAAAGAAAAG                                                                               TTAGATATAC                             GTTTAAAGAA                                                                               TATGTACAAG                                                                               AAAAGAAAGA                                                                               TTTAACTGCT                                                                               GAATTATCTT                             TAAATCTATT                                                                               TATTGATCCT                                                                               ACAACAGTGA                                                                               CGCAAAAAGG                                                                               TAACCAAAAT                             GTTGAAGTTA                                                                               AATTGGGTGA                                                                               GACTACGGTT                                                                               AGCAAAATAT                                                                               TTAATATTCA                             ATATTTAGGT                                                                               GGAGTTAGAG                                                                               ATAATTGGGG                                                                               AGTAACAGCT                                                                               AATGGTCGAA                             TTGATACTTT                                                                               AAATAAAGTA                                                                               GATGGGAAAT                                                                               TTAGTCATTT                                                                               TGCGTACATG                             AAACCTAACA                                                                               ACCAGTCGTT                                                                               AAGCTCTGTG                                                                               ACAGTAACTG                                                                               GTCAAGTAAC                             TAAAGGAAAT                                                                               AAACCAGGGG                                                                               TTAATAATCC                                                                               AACAGTTAAG                                                                               GTATATAAAC                             ACATTGGTTC                                                                               AGACGATTTA                                                                               GCTGAAAGCG                                                                               TATATGCAAA                                                                               GCTTGATGAT                             GTCAGCAAAT                                                                               TTGAAGATGT                                                                               GACTGATAAT                                                                               ATGAGTTTAG                                                                               ATTTTGATAC                             TAATGGTGGT                                                                               TATTCTTTAA                                                                               ACTTTAATAA                                                                               TTTAGACCAA                                                                               AGTAAAAATT                             ATGTAATAAA                                                                               ATATGAAGGG                                                                               TATTATGATT                                                                               CAAATGCTAG                                                                               CAACTTAGAA                             TTTCAAACAC                                                                               ACCTTTTTGG                                                                               ATATTATAAC                                                                               TATTATTATA                                                                               CAAGTAATTT                             AACTTGGAAA                                                                               AATGGCGTTG                                                                               CATTTTACTC                                                                               TAATAACGCT                                                                               CAAGGCGACG                             GCAAAGATAA                                                                               ACTAAAGGAA                                                                               CCTATTATAG                                                                               AACATAGTAC                                                                               TCCTATCGAA                             CTTGAATTTA                                                                               AATCAGAGCC                                                                               GCCAGTGGAG                                                                               AAGCATGAAT                                                                               TGACTGGTAC                             AATCGAAGAA                                                                               AGTAATGATT                                                                               CTAAGCCAAT                                                                               TGATTTTGAA                                                                               TATCATACAG                             CTGTTGAAGG                                                                               TGCAGAAGGT                                                                               CATGCAGAAG                                                                               GTACCATTGA                                                                               AACTGAAGAA                             GATTCTATTC                                                                               ATGTAGACTT                                                                               TGAAGAATCG                                                                               ACACATGAAA                                                                               ATTCAAAACA                             TCATGCTGAT                                                                               GTTGTTGAAT                                                                               ATGAAGAAGA                                                                               TACAAACCCA                                                                               GGTGGTGGTC                             AGGTTACTAC                                                                               TGAGTCTAAC                                                                               CTAGTTGAAT                                                                               TTGACGAAGA                                                                               TTCTACAAAA                             GGTATTGTAA                                                                               CTGGTGCTGT                                                                               TAGCGATCAT                                                                               ACAACAATTG                                                                               AAGATACGAA                             AGAATATACG                                                                               ACTGAAAGTA                                                                               ACTTGATTGA                                                                               ACTAGTAGAT                                                                               GAACTACCTG                             AAGAACATGG                                                                               TCAAGCGCAA                                                                               GGACCAATCG                                                                               AGGAAATTAC                                                                               TGAAAACAAT                             CATCATATTT                                                                               CTCATTCTGG                                                                               TTTAGGAACT                                                                               GAAAATGGTC                                                                               ACGGTAATTA                             TGGCGTGATT                                                                               GAAGAAATCG                                                                               AAGAAAATAG                                                                               CCACGTGGAT                                                                               ATTAAGAGTG                             AATTAGGTTA                                                                               CGAAGGTGGC                                                                               CAAAATAGCG                                                                               GTAATCAGTC                                                                               ATTTGAGGAA                             GACACAGAAG                                                                               AAGATAAACC                                                                               GAAATATGAA                                                                               CAAGGTGGCA                                                                               ATATCGTAGA                             TATCGATTTC                                                                               GATAGTGTAC                                                                               CTCAAATTCA                                                                               TGGTCAAAAT                                                                               AATGGTAACC                             AATCATTCGA                                                                               AGAAGATACA                                                                               GAGAAAGACA                                                                               AACCTAAGTA                                                                               TGAACAAGGT                             GGTAATATCA                                                                               TTGATATCGA                                                                               CTTCGACAGT                                                                               GTGCCACATA                                                                               TTCACGGATT                             CAATAAGCAC                                                                               ACTGAAATTA                                                                               TTGAAGAAGA                                                                               TACAAATAAA                                                                               GATAAACCAA                             ATTATCAATT                                                                               CGGTGGACAC                                                                               AATAGTGTTG                                                                               ACTTTGAAGA                                                                               AGATACACTT                             CCACAAGTAA                                                                               GTGGTCATAA                                                                               TGAAGGTCAA                                                                               CAAACGATTG                                                                               AAGAAGATAC                             AACACCTCCA                                                                               ATCGTGCCAC                                                                               CAACGCCACC                                                                               GACACCAGAA                                                                               GTACCAAGCG                             AGCCGGAAAC                                                                               ACCAACACCA                                                                               CCGACACCAG                                                                               AAGTACCAAG                                                                               CGAGCCGGAA                             ACACCAACAC                                                                               CGCCAACGCC                                                                               AGAGGTACCA                                                                               ACTGAACCTG                                                                               GTAAACCAAT                             ACCACCTGCT                                                                               AAAGAAGAAC                                                                               CTAAAAAACC                                                                               TTCTAAACCA                                                                               GTGGAACAAG                             GTAAAGTAGT                                                                               AACACCTGTT                                                                               ATTGAAATCA                                                                               ATGAAAAGGT                                                                               TAAAGCAGTG                             GTACCAACTA                                                                               AAAAAGCACA                                                                               ATCTAAGAAA                                                                               TCTGAACTAC                                                                               CTGAAACAGG                             TGGAGAAGAA                                                                               TCAACAAACA                                                                               ACGGCATGTT                                                                               GTTCGGCGGA                                                                               TTATTTAGCA                             TTTTAGGTTT                                                                               AGCGTTATTA                                                                               CGCAGAAATA                                                                               AAAAGAATCA                                                                               CAAAGCATAA                             TCAATCCAAA                                                                               ATTGACAGGT                                                                               TTATTTCATA                                                                               AATTATATGA                                                                               AGTAAGCCTG                             TTTTTTAAAA                                                                               TTAAAACAAA                                                                               TTTCCCAAGA                                                                               AATAATTACA                                                                               TACTCAATGA                             CACTATGAAG                                                                               GCGTTCTAAT                                                                               TAGTGTTAAA                                                                               ATGACGTTGA                                                                               TACATAGATT                             TAATACTTAG                                                                               GAAAAGGAGC                                                                               ACATTAACTT                                                                               TGAAAAAAAT                                                                               AAAAAAGGCA                             ATCATTCCCG                                                                               CTGCTGGTTT                                                                               AGGGACTAGA                                                                               TTTTTACCAG                                                                               CAACTAAAGC                             TATCGATTTC                                                                               GATAGTGTAC                                                                               CTCAAATTCA                                                                               TGGTCAAAAT                                                                               AATGGTAACC                             AATCATTCGA                                                                               AGAAGATACA                                                                               GAGAAAGACA                                                                               AACCTAAGTA                                                                               TGAACAAGGT                             GGTAATATCA                                                                               TTGATATCGA                                                                               CTTCGACAGT                                                                               GTGCCACATA                                                                               TTCACGGATT                             CAATAAGCAC                                                                               ACTGAAATTA                                                                               TTGAAGAAGA                                                                               TACAAATAAA                                                                               GATAAACCAA                             ATTATCAATT                                                                               CGGTGGACAC                                                                               AATAGTGTTG                                                                               ACTTTGAAGA                                                                               AGATACACTT                             CCACAAGTAA                                                                               GTGGTCATAA                                                                               TGAAGGTCAA                                                                               CAAACGATTG                                                                               AAGAAGATAC                             AACACCTCCA                                                                               ATCGTGCCAC                                                                               CAACGCCACC                                                                               GACACCAGAA                                                                               GTACCAAGCG                             AGCCGGAAAC                                                                               ACCAACACCA                                                                               CCGACACCAG                                                                               AAGTACCAAG                                                                               CGAGCCGGAA                             ACACCAACAC                                                                               CGCCAACGCC                                                                               AGAGGTACCA                                                                               ACTGAACCTG                                                                               GTAAACCAAT                             ACCACCTGCT                                                                               AAAGAAGAAC                                                                               CTAAAAAACC                                                                               TTCTAAACCA                                                                               GTGGAACAAG                             GTAAAGTAGT                                                                               AACACCTGTT                                                                               ATTGAAATCA                                                                               ATGAAAAGGT                                                                               TAAAGCAGTG                             GTACCAACTA                                                                               AAAAAGCACA                                                                               ATCTAAGAAA                                                                               TCTGAACTAC                                                                               CTGAAACAGG                             TGGAGAAGAA                                                                               TCAACAAACA                                                                               ACGGCATGTT                                                                               GTTCGGCGGA                                                                               TTATTTAGCA                             TTTTAGGTTT                                                                               AGCGTTATTA                                                                               CGCAGAAATA                                                                               AAAAGAATCA                                                                               CAAAGCATAA                             TCAATCCAAA                                                                               ATTGACAGGT                                                                               TTATTTCATA                                                                               AATTATATGA                                                                               AGTAAGCCTG                             TTTTTTAAAA                                                                               TTAAAACAAA                                                                               TTTCCCAAGA                                                                               AATAATTACA                                                                               TACTCAATGA                             CACTATGAAG                                                                               GCGTTCTAAT                                                                               TAGTGTTAAA                                                                               ATGACGTTGA                                                                               TACATAGATT                             TAATACTTAG                                                                               GAAAAGGAGC                                                                               ACATTAACTT                                                                               TGAAAAAAAT                                                                               AAAAAAGGCA                             ATCATTCCCG                                                                               CTGCTGGTTT                                                                               AGGGACTAGA                                                                               TTTTTACCAG                                                                               CAACTAAAGC.                            __________________________________________________________________________


5. A microorganism containing at least a plasmid or phage according to claim
 4. 6. An E. coli strain expressing a fibronectin binding protein encoded by the nucleotide sequence:

    __________________________________________________________________________     GTTAACAACA                                                                               ATCTTAACTT                                                                               TTTATTAACT                                                                               CGCTTTTTTT                                                                               CATTGCTTTT                             AAAAACCGAA                                                                               CAATATAGAA                                                                               TTGCATTTAT                                                                               TGAGTTTTTA                                                                               AAATAAATGA                             ATTTTGCATT                                                                               TAAGGGAGAA                                                                               TATTATAGTG                                                                               AAAAGCAATC                                                                               TTAGATACGG                             CATAAGAAAA                                                                               CACAAATTGG                                                                               GAGCGGCCTC                                                                               AGTATTCTTA                                                                               GGAACAATGA                             TCGTTGTTGG                                                                               AATGGGACAA                                                                               GAAAAAGAAG                                                                               CTGCAGCATC                                                                               GGAACAAAAC                             AATACTACAG                                                                               TAGAGGAAAG                                                                               TGGGAGTTCA                                                                               GCTACTGAAA                                                                               GTAAAGCAAG                             CGAAACACAA                                                                               ACAACTACAA                                                                               ATAACGTTAA                                                                               TACAATAGAT                                                                               GAAACACAAT                             CATACAGCGC                                                                               GACATCAACT                                                                               GAGCAACCAT                                                                               CACAATCAAC                                                                               ACAAGTAACA                             ACAGAAGAAG                                                                               CACCGAAAAC                                                                               TGTGCAAGCA                                                                               CCAAAAGTAG                                                                               AAACTTCGCG                             AGTTGATTTG                                                                               CCATCGGAAA                                                                               AAGTTGCTGA                                                                               TAAGGAAACT                                                                               ACAGGAACTC                             AAGTTGACAT                                                                               AGCTCAACAA                                                                               AGTAAAGTCT                                                                               CAGAAATTAA                                                                               ACCAAGAATG                             AAAAGATCAA                                                                               CTGACGTTAC                                                                               AGCAGTTGCA                                                                               GAGAAAGAAG                                                                               TAGTGGAAGA                             AACTAAAGCG                                                                               ACAGGTACAG                                                                               ATGTAACAAA                                                                               TAAAGTGGAA                                                                               GTAGAAGAAG                             GTAGTGAAAT                                                                               TGTAGGACAT                                                                               AAACAAGATA                                                                               CGAATGTTGT                                                                               AAATCCTCAT                             AACGCAGAAA                                                                               GAGTAACCTT                                                                               GAAATATAAA                                                                               TGGAAATTTG                                                                               GAGAAGGAAT                             TAAGGCGGGA                                                                               GATTATTTTG                                                                               ATTTCACATT                                                                               AAGCGATAAT                                                                               GTTGAAACTC                             ATGGTATCTC                                                                               AACACTGCGT                                                                               AAAGTTCCGG                                                                               AGATAAAAAG                                                                               TACAGATGGT                             CAAGTTATGG                                                                               CGACAGGAGA                                                                               AATAATTGGA                                                                               GAAAGAAAAG                                                                               TTAGATATAC                             GTTTAAAGAA                                                                               TATGTACAAG                                                                               AAAAGAAAGA                                                                               TTTAACTGCT                                                                               GAATTATCTT                             TAAATCTATT                                                                               TATTGATCCT                                                                               ACAACAGTGA                                                                               CGCAAAAAGG                                                                               TAACCAAAAT                             GTTGAAGTTA                                                                               AATTGGGTGA                                                                               GACTACGGTT                                                                               AGCAAAATAT                                                                               TTAATATTCA                             ATATTTAGGT                                                                               GGAGTTAGAG                                                                               ATAATTGGGG                                                                               AGTAACAGCT                                                                               AATGGTCGAA                             TTGATACTTT                                                                               AAATAAAGTA                                                                               GATGGGAAAT                                                                               TTAGTCATTT                                                                               TGCGTACATG                             AAACCTAACA                                                                               ACCAGTCGTT                                                                               AAGCTCTGTG                                                                               ACAGTAACTG                                                                               GTCAAGTAAC                             TAAAGGAAAT                                                                               AAACCAGGGG                                                                               TTAATAATCC                                                                               AACAGTTAAG                                                                               GTATATAAAC                             ACATTGGTTC                                                                               AGACGATTTA                                                                               GCTGAAAGCG                                                                               TATATGCAAA                                                                               GCTTGATGAT                             GTCAGCAAAT                                                                               TTGAAGATGT                                                                               GACTGATAAT                                                                               ATGAGTTTAG                                                                               ATTTTGATAC                             TAATGGTGGT                                                                               TATTCTTTAA                                                                               ACTTTAATAA                                                                               TTTAGACCAA                                                                               AGTAAAAATT                             ATGTAATAAA                                                                               ATATGAAGGG                                                                               TATTATGATT                                                                               CAAATGCTAG                                                                               CAACTTAGAA                             TTTCAAACAC                                                                               ACCTTTTTGG                                                                               ATATTATAAC                                                                               TATTATTATA                                                                               CAAGTAATTT                             AACTTGGAAA                                                                               AATGGCGTTG                                                                               CATTTTACTC                                                                               TAATAACGCT                                                                               CAAGGCGACG                             GCAAAGATAA                                                                               ACTAAAGGAA                                                                               CCTATTATAG                                                                               AACATAGTAC                                                                               TCCTATCGAA                             CTTGAATTTA                                                                               AATCAGAGCC                                                                               GCCAGTGGAG                                                                               AAGCATGAAT                                                                               TGACTGGTAC                             AATCGAAGAA                                                                               AGTAATGATT                                                                               CTAAGCCAAT                                                                               TGATTTTGAA                                                                               TATCATACAG                             CTGTTGAAGG                                                                               TGCAGAAGGT                                                                               CATGCAGAAG                                                                               GTACCATTGA                                                                               AACTGAAGAA                             GATTCTATTC                                                                               ATGTAGACTT                                                                               TGAAGAATCG                                                                               ACACATGAAA                                                                               ATTCAAAACA                             TCATGCTGAT                                                                               GTTGTTGAAT                                                                               ATGAAGAAGA                                                                               TACAAACCCA                                                                               GGTGGTGGTC                             AGGTTACTAC                                                                               TGAGTCTAAC                                                                               CTAGTTGAAT                                                                               TTGACGAAGA                                                                               TTCTACAAAA                             GGTATTGTAA                                                                               CTGGTGCTGT                                                                               TAGCGATCAT                                                                               ACAACAATTG                                                                               AAGATACGAA                             AGAATATACG                                                                               ACTGAAAGTA                                                                               ACTTGATTGA                                                                               ACTAGTAGAT                                                                               GAACTACCTG                             AAGAACATGG                                                                               TCAAGCGCAA                                                                               GGACCAATCG                                                                               AGGAAATTAC                                                                               TGAAAACAAT                             CATCATATTT                                                                               CTCATTCTGG                                                                               TTTAGGAACT                                                                               GAAAATGGTC                                                                               ACGGTAATTA                             TGGCGTGATT                                                                               GAAGAAATCG                                                                               AAGAAAATAG                                                                               CCACGTGGAT                                                                               ATTAAGAGTG                             AATTAGGTTA                                                                               CGAAGGTGGC                                                                               CAAAATAGCG                                                                               GTAATCAGTC                                                                               ATTTGAGGAA                             GACACAGAAG                                                                               AAGATAAACC                                                                               GAAATATGAA                                                                               CAAGGTGGCA                                                                               ATATCGTAGA                             TATCGATTTC                                                                               GATAGTGTAC                                                                               CTCAAATTCA                                                                               TGGTCAAAAT                                                                               AATGGTAACC                             AATCATTCGA                                                                               AGAAGATACA                                                                               GAGAAAGACA                                                                               AACCTAAGTA                                                                               TGAACAAGGT                             GGTAATATCA                                                                               TTGATATCGA                                                                               CTTCGACAGT                                                                               GTGCCACATA                                                                               TTCACGGATT                             CAATAAGCAC                                                                               ACTGAAATTA                                                                               TTGAAGAAGA                                                                               TACAAATAAA                                                                               GATAAACCAA                             ATTATCAATT                                                                               CGGTGGACAC                                                                               AATAGTGTTG                                                                               ACTTTGAAGA                                                                               AGATACACTT                             CCACAAGTAA                                                                               GTGGTCATAA                                                                               TGAAGGTCAA                                                                               CAAACGATTG                                                                               AAGAAGATAC                             AACACCTCCA                                                                               ATCGTGCCAC                                                                               CAACGCCACC                                                                               GACACCAGAA                                                                               GTACCAAGCG                             AGCCGGAAAC                                                                               ACCAACACCA                                                                               CCGACACCAG                                                                               AAGTACCAAG                                                                               CGAGCCGGAA                             ACACCAACAC                                                                               CGCCAACGCC                                                                               AGAGGTACCA                                                                               ACTGAACCTG                                                                               GTAAACCAAT                             ACCACCTGCT                                                                               AAAGAAGAAC                                                                               CTAAAAAACC                                                                               TTCTAAACCA                                                                               GTGGAACAAG                             GTAAAGTAGT                                                                               AACACCTGTT                                                                               ATTGAAATCA                                                                               ATGAAAAGGT                                                                               TAAAGCAGTG                             GTACCAACTA                                                                               AAAAAGCACA                                                                               ATCTAAGAAA                                                                               TCTGAACTAC                                                                               CTGAAACAGG                             TGGAGAAGAA                                                                               TCAACAAACA                                                                               ACGGCATGTT                                                                               GTTCGGCGGA                                                                               TTATTTAGCA                             TTTTAGGTTT                                                                               AGCGTTATTA                                                                               CGCAGAAATA                                                                               AAAAGAATCA                                                                               CAAAGCATAA                             TCAATCCAAA                                                                               ATTGACAGGT                                                                               TTATTTCATA                                                                               AATTATATGA                                                                               AGTAAGCCTG                             TTTTTTAAAA                                                                               TTAAAACAAA                                                                               TTTCCCAAGA                                                                               AATAATTACA                                                                               TACTCAATGA                             CACTATGAAG                                                                               GCGTTCTAAT                                                                               TAGTGTTAAA                                                                               ATGACGTTGA                                                                               TACATAGATT                             TAATACTTAG                                                                               GAAAAGGAGC                                                                               ACATTAACTT                                                                               TGAAAAAAAT                                                                               AAAAAAGGCA                             ATCATTCCCG                                                                               CTGCTGGTTT                                                                               AGGGACTAGA                                                                               TTTTTACCAG                                                                               CAACTAAAGC.                            __________________________________________________________________________ 